The objective of this study was to statistically optimize the mineral components of the nutritional
medium required for enhancing the production of a cold-active extracellular serine-type protease, W-Pro21717,
by the Antarctic bacterium Pseudoalteromonas arctica PAMC 21717. Skim milk was identified as
the major efficient inducer. Among the 12 components included in the unoptimized medium, skim milk,
NaCl, Na2SO4, Fe(C6H5O7) (ferric citrate), and KCl were determined, by the Plackett？Burman and Box？
Behnken design, to have a major effect on W-Pro21717 production. Fed-batch fermentation (5 L scale)
using the mineral-optimized medium supplemented with concentrated skim milk (critical medium
component) resulted in a W-Pro21717 activity of 53.4 U/L, a 15-fold increment in production over that
obtained using unoptimized flask culture conditions. These findings could be applied to scale up the
production of cold-active protease.